Project Summary Cytomegalovirus (CMV) is a leading cause of oral ulcerations in immunocompromised individuals and is a factor in periodontal disease. There may also be a link between CMV infection and cleft lip/palate development. Saliva is a major route of transmission for the virus. Infecting over half of the adult population worldwide, CMV is consequently a major oral health concern. The broad tissue tropism of CMV is one reason the virus causes severe illness in these tissues. However, much is still not understood about what dictates the ability of the virus to enter various cell types. Entry receptors for a virus are a major determinant of viral tropism. While much work has been done on CMV entry into fibroblasts, the identity of the entry receptor used for infection of epithelial and endothelial cells has remained unknown until just in the last year Neuropilin-2 was put forward. We have independently identified Neuropilin-1 (Nrp-1) as a putative entry receptor for murine CMV (MCMV). No entry receptor for MCMV has been proposed before now. Using MCMV to study the details of Nrp-1-dependent infection will provide insights into a key CMV-host interaction that may be targeted to block infection and reduce infections in the mouth. No role for Nrp-1 in MCMV infection has been reported previously. Preliminary data points to Nrp-1 as mediating viral entry. This will first be investigated more closely by assessing colocalization of Nrp-1 and MCMV during infection, measuring binding between virus and Nrp-1, and quantifying viral internalization in the absence of Nrp-1. Next, the mode of entry regulated by Nrp-1 will be determined using inhibitors of endocytic pathways and comparing markers of these pathways during infection with and without Nrp-1. Taking advantage of MCMV as a natural mouse pathogen, we will also test whether Nrp-1 is required for infection and spread to the salivary glands in vivo. Lastly, the mechanics of the interaction between the virus and Nrp-1 will be examined. The viral protein bound by Nrp-1 will be identified by immunoprecipitations and mass spectrometry. Additionally, the region of Nrp-1 that binds to the virus will be located. Mutant Nrp-1 constructs will be generated and used to reconstitute Nrp-1 knockout cells. The ability of each of these mutants to allow infection will then be determined. This information will be critical for the design of a therapy that targets the receptor-binding activity of CMV.